The molecular determinants of albumin permeability in the vascular endothelium have profound effects on capillary solute and water exchange and delivery of bloodborne substances. Based on conventional imaging in fixed tissue and culture cells, it is apparent that a receptor (gp60) mediated transcytosis of native albumin occurs within the pulmonary circulation and this process is an important component of overall pulmonary vascular permeability. S-nitrosoalbumin (SNO-Alb) is considered as the most abundant physiological stable circulating NO carrier in human plasma with vasodilatory and antiaggregation properties similar to NO. In light of the importance of receptor mediated transcytosis of native albumin, it is noteworthy that there is no information on the disposition and effect of SNO-Alb within the pulmonary endothelium. We hypothesize that SNO-Alb will be transported by similar receptor-mediated caveolae-associated transcytosis as native albumin, and then, by serving as an intracellular source of NO, will potentially modulate endothelial cell metabolism and permeability via NO release (and activation of soluble guanylyl cyclase) and/or S(trans)-nitrosation. We propose to use a comprehensive multimode imaging approach involving full spectral confocal and multiiphoton laser scanning and total internal reflection fluorescence microscopy to determine the nature of SNO-albumin transport in live cultured rat pulmonary microvascular endothelium (RPVEC) and isolated perfused rat lungs (IPL). Accordingly, the specific aims of this proposal are to: 1} identify the cellular mechanisms by which SNO-albumin is transported across RPVEC and mechanism of transport of SNO-albumin in the intact pulmonary endothelium of IPL; 2) identify signaling pathways activated by SNO-alb transport within pulmonary endothelium; 3) Determine the effect of SNO-Alb on permeability in RPVEC and in rat IPL; 4) Determine the spectrum of proteins that are S-nitrosated by SNO-albumin via 2D gel differential fluorescence gel electrophoresis, biotin switch and MALTI TOF mass spectrometry.